CN105288702B - A kind of acellular polysaccharide dermal matrix material and its preparation method and application - Google Patents

Abstract

The invention relates to a cell-free polysaccharide corium matrix material and its preparation method and application. The material includes cell-free dermis matrix material and polysaccharide, and the polysaccharide solution is a composite solution of carboxymethyl chitosan, chondroitin sulfate and hyaluronic acid. The preparation method of the acellular polysaccharide dermal matrix material is as follows: select the PADM after packaging and disinfection, wash by weighing, compounding carboxymethyl chitosan, compounding chondroitin sulfate, glutaraldehyde cross-linking, compounding hyaluronic acid, and PBS buffer solution. , packaging, sterilization, etc. The functional material added to the cell-free polysaccharide dermal matrix material can be used to prepare biological dressings, composite transplantation brackets, and tissue engineering epidermal brackets. The acellular polysaccharide dermal matrix material has good in vitro degradability, high tensile strength, high water vapor permeability, high porosity and excellent antibacterial performance.

Description

A kind of acellular polysaccharide dermal matrix material and its preparation method and application

technical field

The invention mainly relates to the field of bioengineering, in particular to a cell-free polysaccharide dermis matrix material and its preparation and application.

Background technique

Acellular matrix (acellular dermal matrix, ADM) is obtained by using physical, chemical, biological and other methods to completely remove the epidermis and cells containing antigen components in the skin, leaving only the extracellular matrix containing collagen in the dermis. It has resistogenicity, good biological properties, and good mechanical properties. It is an ideal biomaterial. Compared with allogenic acellular dermal matrix, xenogeneic (pig) acellular dermal matrix has the advantages of wide source, low price and high quality. It has high tissue homology and is highly similar to human skin in terms of content and weaving structure. After modification and cross-linking, its antigenicity can be further reduced, mechanical properties can be improved, and the antibacterial property of the material can be increased, which has great application prospects.

Chitin is a widely distributed and easy-to-extract polymer, which exists in the shells of crustaceans such as shrimps and crabs, as well as in the cell walls of bacteria, insects, algae, and higher plants. Chitosan is the deacetylation degree of chitin at 60 The product above % is a linear, semi-crystalline polysaccharide. Carboxymethyl chitosan is a derivative of chitosan. By introducing small chemical groups such as carboxymethyl, it has Good water solubility without affecting the inherent properties of chitosan. Compared with chitosan, carboxymethyl chitosan has better water retention, biocompatibility, adhesion, antibacterial, biodegradability and low toxicity, and carboxymethyl chitosan can accelerate fibroblast Proliferation and promotion of wound healing, these excellent properties make carboxymethyl chitosan more suitable as a biomedical wound repair material and has broad application prospects.

Chondroitin sulfate (CS) is a kind of glycosaminoglycan covalently linked to protein to form proteoglycan, which is widely distributed in the extracellular matrix and cell surface of animal tissues, and has anticoagulant effect. Compounding it on the surface of the matrix can increase the biocompatibility of the dressing, facilitate cell adhesion and proliferation, and accelerate wound healing.

Hyaluronic acid, also known as hyaluronic acid and HA, is an acidic mucopolysaccharide, which belongs to a high molecular polymer. It is composed of N-acetylglucosamine and D-glucuronic acid through glycosidic bonds. This polysaccharide has high water retention. It can maintain cell moisture, protect cells from pathogenic bacteria, and prevent infection. At the same time, it can speed up the recovery of skin tissue and improve the ability of wound healing and regeneration. Compounding hyaluronic acid on the matrix can improve the wound repair ability of the dressing, accelerate epithelialization, regulate collagen synthesis, effectively reduce scar formation, and make the healed tissue closer to normal skin.

Compared with synthetic materials, biomaterials have the advantages of good biocompatibility and easy cell attachment and growth, but they also have the disadvantages of fast degradation rate, difficult control of microstructure, and insufficient mechanical strength. For example, patent CN1387923A discloses a heterogeneous acellular dermal scaffold and its preparation method. The scaffold is obtained by treating suckling pigskin with trypsin and glutaraldehyde. Capacitance is better, but its permeability is poor, and the wound surface is prone to fluid accumulation and gas accumulation, and the vascularization is slow. Patent CN1343523A discloses a microporous allogeneic or heterogeneous acellular dermal substitute that can be used for wound repair, using mechanical or laser methods to regularly make dense penetrating holes on the acellular dermis, although this can solve the problem of acellular The disadvantage of poor permeability of dermal substitutes, but too large or too dense pore size will increase the exposure of antigenic sites and the chance of antigen-presenting cells contacting ADM, resulting in poor cell compatibility and destroying the collagen scaffold to a certain extent. structure. Patent CN1775189A discloses an acellular dermal matrix and its preparation method. The acellular dermal matrix is a product obtained by sequentially treating mammalian skin with sodium hydroxide solution and detergent. The acellular dermal matrix treated by this method has the advantages of good histocompatibility and good degradability, but Its tensile strength needs to be improved, and its antibacterial performance is insufficient.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art, and provide a method for preparing an acellular polysaccharide dermal matrix material with low antigenicity, excellent biocompatibility, good mechanical properties and excellent antibacterial properties.

A cell-free polysaccharide dermal matrix material: water-soluble polysaccharides are compounded on the acellular polysaccharide dermal matrix, and the water-soluble polysaccharides are composed of carboxymethyl chitosan, chondroitin sulfate, and hyaluronic acid.

For the first time, it adopts the method of combining carboxymethyl chitosan, chondroitin sulfate, and hyaluronic acid. The preparation method of a cell-free polysaccharide dermal matrix material specifically includes the following steps:

(1) select porcine acellular dermal matrix (PADM) through sterilized packaging, and accurately weigh as the basis for counting the following procedures;

(2) Composite carboxymethyl chitosan: Put the prepared PADM into the drum, the mass ratio of PADM to PADM material is 1.5-3.0:1, the temperature is 25-45°C, the pH value is 5.5-6.0, and the mass concentration is Add 0.50-1.20% carboxymethyl chitosan solution to the drum in stages, adjust the pH value to 4.5-5.5 after reacting for 30 minutes, and adjust the pH value to 6.5-7.0 after reacting for 60-90 minutes. Clean the waste liquid, and then wash the treated material with distilled water with a mass ratio of 4.0:1 for 2 times, each time for 15 minutes, and drain the water after washing;

(3) Glutaraldehyde cross-linking: at a temperature of 25°C and a pH value of 3.0 to 5.0, add 0.1 to 0.20% glutaraldehyde to the material obtained in step (2) with a mass ratio of 2.0:1 to the PADM material Add the solution in stages, gradually adjust the pH value to 8.0, react for 30-60 minutes, take out the material, wash with distilled water until the pH value is 7.0-7.3;

(4) Composite chondroitin sulfate: add the chondroitin sulfate solution with a mass ratio of 1.5 to 3.0:1, a temperature of 25 to 45°C, and a mass concentration of 0.80 to 1.00% with the PADM material, and add the product of step (3) in batches. In the drum, after reacting for 60 minutes, adjust the pH value to 6.0-7.0, and then wash the treated material with distilled water with a mass ratio of 2.0:1 for 2 times, each time for 15 minutes, and drain the water after washing;

(5) Composite hyaluronic acid: fully dissolve 1.00g hyaluronic acid in acetic acid solution, dilute it to 100ml, and make a solution with a concentration of 1.00% (w/v), and compound carboxymethyl chitosan, sulfuric acid The acellular dermal matrix material after chondroitin is centrifugally dehydrated so that its volume water content is 70-90%, and then the hyaluronic acid solution is sprayed onto the surface of the acellular dermal matrix material with a small spray bottle, and the spraying amount is 0.5-1g/m ² ;

(6) Washing with PBS solution: at a temperature of 25° C., rinse with PBS solution with a mass ratio of 3.0:1 to the above-mentioned materials for 3 times, each time for 30 minutes.

(7) Encapsulation: the material is bagged and sealed;

(8) Disinfection: The packaged materials were irradiated with cobalt-60 100Gy for disinfection, and stored in a sterile environment at -4°C.

Application of the above-mentioned acellular polysaccharide dermal matrix material: adding functional materials to the material can be used to prepare biological dressings, composite graft scaffolds, and tissue engineering epidermal scaffolds, wherein the functional materials are antibacterial agents, antifungal agents, wound healing agents, Growth factors, skin basement membrane formation promoters, glycerin. For example, adding glycerin with a volume content of 1.0-3.0% to the material can make an antibacterial biological dressing, and sticking one side of it to the wound can serve the medical purpose of protecting the wound and absorbing body fluids. For example, adding 3-6 μg/ ^m2 of growth factors to the acellular polysaccharide dermal matrix material, 8-12 μg/ ^m2 of skin basement membrane formation promoters, and 1.0-3.0% of glycerin can make an artificial epidermis with antibacterial effects stand.

The acellular dermal matrix material has strong tensile strength, which provides a suitable growth space for cells and is conducive to the natural growth and arrangement of cells. Carboxymethyl chitosan has good water retention, biocompatibility, adhesion Antibacterial, antibacterial, biodegradable and low toxicity, while carboxymethyl chitosan can accelerate fibroblast proliferation and promote wound healing. Chondroitin sulfate (CS) has an anticoagulant effect, and compounding it on the surface of the matrix can increase the biocompatibility of the material, facilitate cell adhesion and proliferation, and accelerate wound healing. Hyaluronic acid has high water retention, which can keep cells hydrated, protect cells from pathogenic bacteria, and prevent infection. At the same time, it can speed up the recovery of skin tissue and improve the ability of wound healing and regeneration. Compounding hyaluronic acid on the matrix can improve the wound repair ability of the material, accelerate epithelialization, regulate collagen synthesis, effectively reduce scar formation, and make the healed tissue closer to normal skin.

Compared with the prior art, the present invention has the following advantages:

1. The acellular polysaccharide dermal matrix material has good degradability. Experiments show that the amount of carboxymethyl chitosan has a certain influence on the in vitro degradation time of the dressing. With the increase of the amount of carboxymethyl chitosan, the in vitro degradation time of the dressing increases, and the material is completely degradable.

2. The acellular polysaccharide dermal matrix material has good water vapor permeability. Experiments show that as the amount of carboxymethyl chitosan increases, the water vapor permeability of the dressing increases, and the use of carboxymethyl chitosan improves the water vapor permeability of the material. The data show that the amount of carboxymethyl chitosan is between 0.00 and 1.50 %, the water vapor transmission rate of the material is between 3400 and 3700g.m ^-2 .24h ^-1 , and the water vapor transmission rate is obviously improved.

3. The acellular polysaccharide dermal matrix material has the advantage of high porosity. As the amount of carboxymethyl chitosan increases, the porosity of the dressing increases. When the amount of carboxymethyl chitosan is 0.00-1.50%, the porosity of the material is between 73-82%. has been significantly enhanced.

4. The acellular polysaccharide dermal matrix material has excellent tensile strength. With the increase of the amount of carboxymethyl chitosan, the tensile strength of the dressing was obviously enhanced. When the amount of carboxymethyl chitosan is 0.00-1.50%, the tensile strength of the material is between 9.40-10.80 MPa.

5. The acellular polysaccharide dermal matrix material has high antibacterial properties. Experiments show that the antibacterial rate of materials without carboxymethyl chitosan is 3.10%. When the material is compounded with 1.00% carboxymethyl chitosan, the antibacterial rate of the material is greater than 70%, indicating that the material has good antibacterial properties.

6. The acellular polysaccharide dermal matrix material has broad applications. We can prepare acellular polysaccharide dermal matrix materials into biological dressings, composite graft scaffolds, and tissue engineering epidermal scaffolds according to different requirements.

Description of drawings

Fig. 1 is a production process flow chart of the present invention;

Figure 2 is the dressing grain map SEM × 500;

Figure 3 is the dressing grain map SEM×1000.

Detailed ways

The present invention is specifically described below by way of examples. Example 1:

This embodiment provides a method for preparing a cell-free polysaccharide dermal matrix dressing, which is prepared by the following steps:

(1) select porcine acellular dermal matrix (PADM) through sterilized packaging, and accurately weigh as the basis for counting the following procedures;

(2) Composite carboxymethyl chitosan: Put the prepared acellular dermal matrix into the drum, and add PADM material with a mass ratio of 2.5:1 at a temperature of 40°C and a pH value of 6.0. The mass concentration is 1.00% carboxymethyl chitosan solution, the pH value is adjusted to 5.0 after 30 minutes of reaction, and the pH value is adjusted to 7.0 after another 60 minutes of reaction. Wash the treated material twice with distilled water, 15 minutes each time, and drain the water after washing;

(3) Glutaraldehyde cross-linking: Add 0.20% glutaraldehyde with a mass ratio of 2.0 to the PADM material under the condition of a temperature of 25°C and a pH value of 5.0, and add it to the drum with the product of step (2) in stages, gradually Adjust the pH value to 8.0, react for 30 minutes, take out the material, wash with distilled water until the pH value is 7.0-7.3;

(4) Composite chondroitin sulfate: at a temperature of 36°C and a pH value of 7.5, add a 1.00% chondroitin sulfate solution with a mass ratio of 2.5:1 to the PADM material, and add the product of step (3) in batches. In a drum, after reacting for 60 minutes, adjust the pH to 7.0. The processed material was washed twice with distilled water, 15 minutes each time, and dried until no water dripped after washing;

(5) Composite hyaluronic acid: fully dissolve 1.00 g of hyaluronic acid in acetic acid solution, dilute to 100 ml, and prepare a solution with a concentration of 1.00% (w/v). The cell-free dermal matrix compounded with carboxymethyl chitosan and chondroitin sulfate is centrifuged and dehydrated to make its volume water content about 80%. Then spray the hyaluronic acid solution onto the surface of the acellular dermal matrix with a small spray bottle, and the spraying amount is 1g/m ² ;

(6) Washing with PBS solution: at a temperature of 25° C., wash with distilled water with a mass ratio of 3.0 to the above materials for 3 times, each time for 30 minutes.

(7) Encapsulation: the material is bagged and sealed;

(8) Disinfection: sterilize the encapsulated material with a dose of cobalt-60 100Gy, and store it in a sterile environment at -4°C;

The antibacterial rate of the acellular polysaccharide dermal matrix material prepared by the above method is greater than 70%, the tensile strength is greater than 10MPa, the water vapor transmission rate is greater than 3600g.m ^-2 .24h ^-1 , and the porosity is greater than 80%. The above data show that the material has good antibacterial effect, excellent mechanical properties, high water vapor permeability, high porosity, and good biocompatibility.

Adding 2wt.% glycerin to the material can make an antibacterial biological dressing, and sticking one side of the dressing on the wound can serve the medical purposes of protecting the wound and absorbing body fluids.

The dressing was observed by SEM, and the results are shown in Figure 2 and Figure 3:

We can see from the figure that the collagen fibers of this acellular polysaccharide dermal matrix antibacterial dressing are arranged in a network structure, the structure is loose, and there are a large number of holes distributed, which is conducive to the volatilization of water vapor and makes the dressing have good water and vapor permeability. . In addition, the thickness of the collagen fibers is uneven, the interstitium of the collagen fibers is removed, and there are large pores between the fibers, which makes the dressing conducive to the growth of tissue cells and vascularization, and promotes wound healing.

Test the tensile strength of the dressing, refer to GB/T1040.3-2006, the tensile strength is the tensile strength per unit cross-sectional area, the calculation formula is:

T _S =F/S

In the formula: TS-tensile strength (MPa), F-the maximum tensile force (N) of the sample when it breaks, S-the cross-sectional area of the sample when it breaks (mm ² ). The sample is measured under the same water content, the chuck separation speed is about 100±10mm per minute, and the sample does not shift during the stretching process.

The result is as follows:

Table 1 Tensile strength of acellular polysaccharide dermal matrix antibacterial dressings

The results show that this acellular polysaccharide dermal matrix antibacterial dressing has good tensile strength, that is, it has good mechanical properties.

The water vapor transmission rate of the dressing is tested by using the cup water method similar to the Aiba method. Take a test tube with a diameter of 2 cm and fill it with physiological saline (0.9%), so that the film is in contact with the liquid surface, leaving no gaps in the middle, and along the side of the test tube. Seal and weigh, put the system in a desiccator at 36±1°C, weigh after 24 hours, and calculate the water vapor transmission rate according to the following formula:

Wr=(M ₀ -M ₁ )/S

In the formula, Wr: water vapor permeability (g·m ^-2 ·24h ^-1 ); M ₀ : original weight (g); M ₁ : weight after 24 hours (g); S: area of test tube mouth (m ² )

The result is as follows:

Table 2 Water vapor transmission rate of acellular polysaccharide dermal matrix antibacterial dressing

The experimental results show that this acellular polysaccharide dermal matrix antibacterial dressing has excellent water vapor permeability.

To test the porosity of the dressing, freeze-dry the sample at -20°C, weigh 5-6g (W ₁ ), accurate to 0.001g, put it into a 50ml volumetric flask, and add toluene to the volumetric flask to the mark. Circulate the vacuum until the sponge no longer has air bubbles overflowing, weigh the volumetric flask (W ₂ ) that still has toluene and the support material, then take out the support material containing toluene inside, and put the remaining toluene and the volumetric flask (W- ₃ ). Calculate the porosity (P) according to the following formula:

P=(W ₂ -W ₃ -W ₁ )/(W ₂ -W ₃ )×100%

The result is as follows:

Table 3 Porosity of acellular polysaccharide dermal matrix antibacterial dressings

Experimental results show that the acellular polysaccharide dermal matrix antibacterial dressing has high porosity, which is conducive to the passage of water vapor.

To test the antibacterial performance of the dressing, cut the acellular polysaccharide dermal matrix antibacterial dressing into a size of 2.0cm×2.0cm, place it in a sterile Erlenmeyer flask as a sample group, and use the polysaccharide-free matrix dressing as a control sample group according to the above method. At the same time prepare no sample group. Add 70 mL of phosphate buffer (0.03 mol·L ^-1 ) and 5 mL of Staphylococcus aureus (or Candida albicans) bacterial solution to the three groups respectively, and shake for 1 hour at 25°C and 200 r·min ^-1 . Take 100ul drops of the plate at 0h and 1h respectively, after the liquid penetrates into the medium, culture at 37°C for 12-24 hours, and count the colonies. Bacteriostatic rate calculation formula:

X=(A－B)/A×100%

X is the bacteriostatic rate, A is the average number of colonies before shaking the sample, and B is the average number of colonies after shaking the sample.

Judgment criteria: the difference in the number of colonies before and after shaking in the group without samples must be within 10%, and the difference between the bacteriostatic rate of the test sample and the control sample is ≥ 26%, which can be judged that the product has antibacterial effect.

The result is as follows:

Table 4 The antibacterial rate of acellular polysaccharide dermal matrix antibacterial dressing

Experimental results show that this acellular polysaccharide dermal matrix antibacterial dressing has excellent antibacterial properties.

Example 2:

This embodiment provides a method for preparing an acellular polysaccharide dermal matrix-epidermal scaffold, which takes the following steps:

(1) Prepare the acellular polysaccharide dermal matrix material according to the operation of Example 1;

(2) Composite 5 μg/m ² of growth factors, 10 μg/m ² of skin basement membrane formation accelerator, and 2.0% glycerin on the surface of the material.

The preparation of the epidermal scaffold by the method can protect the wound, promote the growth of the skin, and improve the healing and regeneration ability of the wound.

Claims (1)

1. a preparation method of acellular polysaccharide dermal matrix, is characterized in that: compound water-soluble polysaccharide on acellular dermal matrix, described water-soluble polysaccharide comprises three kinds of carboxymethyl chitosan, chondroitin sulfate, hyaluronic acid jointly constituted; Concrete preparation method step is: (1) select the porcine acellular dermal matrix PADM through sterilized packaging, and accurately weigh it as the basis for counting the following procedures; (2) Composite carboxymethyl chitosan: put the prepared PADM into the drum, and put the carboxymethyl chitosan with a mass ratio of 1.5-3.0:1, a temperature of 25-45°C, and a pH value of 5.5-6.0 to the PADM material. Add the chitosan solution to the drum in stages, adjust the pH value to 4.5-5.5 after reacting for 30 minutes, and adjust the pH value to 6.5-7.0 after reacting for 60-90 minutes. The material was washed twice with distilled water with a mass ratio of 4.0:1, each time for 15 minutes, and the water was drained after washing; (3) Glutaraldehyde cross-linking: at a temperature of 25°C and a pH value of 3.0 to 5.0, add 0.1 to 0.20% glutaraldehyde to the material obtained in step (2) with a mass ratio of 2.0:1 to the PADM material Add the solution in stages, gradually adjust the pH value to 8.0, react for 30-60 minutes, take out the material, wash with distilled water until the pH value is 7.0-7.3; (4) Composite chondroitin sulfate: add the chondroitin sulfate solution with a mass ratio of 1.5 to 3.0:1 to PADM material and a temperature of 25 to 45°C into the drum containing the product of step (3) in batches, and react for 60 minutes Finally, adjust the pH value to 6.0-7.0, and then wash the treated material with distilled water with a mass ratio of 2.0:1 for 2 times, each time for 15 minutes, and dry the water after washing; (5) Composite hyaluronic acid: fully dissolve hyaluronic acid in acetic acid solution to form a hyaluronic acid solution, and centrifugally dehydrate the acellular dermal matrix material compounded with carboxymethyl chitosan and chondroitin sulfate to make it The volume water content is 70-90%, and then spray the hyaluronic acid solution on the surface of the acellular dermal matrix material with a small spray bottle; (6) Washing with PBS solution: at a temperature of 25°C, wash with PBS solution with a mass ratio of 3.0:1 to the material obtained above for 3 times, each time for 30 minutes; (7) Encapsulation: the material is bagged and sealed; (8) Disinfection: sterilize the encapsulated material with a dose of cobalt-60 100Gy, and store it in a sterile environment at -4°C; In step (2), the carboxymethyl chitosan mass concentration is between 0.50% and 1.20%; In step (3), the mass concentration of glutaraldehyde is between 0.1% and 0.2%; In step (4), the mass concentration of the chondroitin sulfate is between 0.80% and 1.00%; In step (5), the concentration of the hyaluronic acid solution is 1.00% w/v solution, and the spraying amount of the hyaluronic acid solution is 0.5-1.0 g/m ² .